We demonstrated that an additional function of the wtRep68 was its ability to bind to a linear form of the ITR, delta57 ITR, with the same affinity as to wt hairpin ITR (Chiorini et al. J. Virol. 1994). A dissociation constant of approximately 8 x 10-10 M was determined for the wtITR and delta57 ITR probes with MBP-Rep68delta. However, truncation of delta57 ITR to delta28 ITR, or cleavage with DdeI inhibited binding by at least 10-fold. Footprinting analysis indicated that the DdeI truncation cuts within the protected region. Extension of delta28 ITR with random sequence restored the ability of MBP-Rep68delta to bind to delta28 ITR. Thus the increase in binding would appear to be the result of non-specific sequence stabilization of MBP-Rep68delta binding. To further define the MBP-Rep68delta binding site, a series of oligonucleotide probes were constructed which spanned the region protected by MBP-Rep78 in a DNase 1 footprinting experiment. Two 18 bp probes which contained the imperfect (GCTC)4 repeat motif were found to be sufficient for the binding. While the above data shows that Rep68 is able to bind to non-hairpin derivatives of the ITR and identifies some of the features required for binding of Rep68 to DNA, this ability of Rep68 to bind to linear DNA does not appear to correlate with terminal resolution site endonuclease activity. The covalent linkage of Rep68 to the non-hairpin containing ITR, delta57 ITR, is approximately 100-fold less efficient compared to linkage to a hairpin-containing ITR. This effect of the hairpin portion of the ITR was observed only when the hairpin was part of the probe and not when it was supplied in trans.